Coral hericium erinaceus with antioxidant activity as well as culture method and application of coral hericium erinaceus

Abstract

The invention provides coral hericium erinaceus with antioxidant activity and a culture method and application thereof, and relates to the technical field of microbial fermentation. According to the method, the schisandra chinensis is adopted as a substrate for bidirectional fermentation with the coral hericium erinaceus for the first time, the coral hericium erinaceus rich in gamma-aminobutyric acid and related products of the coral hericium erinaceus are obtained, and reference is provided for developing the coral hericium erinaceus into a functional food base material and related antioxidant food.

Description

Technical Field

[0001] The present application relates to the field of microbial fermentation technology, and in particular to a coral hericium with antioxidant activity and a cultivation method and application thereof. Background Art

[0002] Hericium coralloides, also known as Jade Beard, belongs to the Basidiomycetes, Agaricomycetes, Russulales, Hericiaceae, Hericium, and is a famous rare and precious edible and medicinal fungus in my country. Hericium coralloides is beneficial to the five internal organs, nourishing, and aiding digestion. It is mainly used for diseases such as neurasthenia and gastric ulcer. It is an excellent medicinal fungus with extremely high nutritional value and health value. The main active ingredient of Hericium erinaceus is polysaccharide. In addition to polysaccharides, it also contains oligosaccharides, sterols, hericin and hericinone and other bioactive substances. It has multiple functions such as improving immunity, anti-tumor, lowering blood sugar, anti-mutation, anti-aging, etc., and has been widely used in medical and other industries.

[0003] γ-aminobutyric acid (GABA) is a non-protein amino acid widely found in animals, plants and microorganisms. Its molecular formula is C4H9NO2. It was first discovered in potato tubers by Steward et al. in 1949. As an important endogenous inhibitory neurotransmitter in the mammalian brain, GABA plays a vital role in maintaining the balance between excitation and inhibition of neuronal networks. In addition, GABA also has physiological activities in animals, such as regulating blood pressure, anti-aging, improving liver and kidney function, regulating the executive ability of bipolar disorder, and assisting the body in maintaining immune homeostasis. Because GABA has important physiological functions for the human body, it is often added to food, nutritional supplements and medicines. The metabolism of GABA in healthy adults is balanced, but aging and increased stress may affect the normal accumulation of GABA, leading to symptoms such as anxiety, fatigue and restlessness, and it needs to be supplemented from food.

[0004] However, there is no precedent for using coral hericium to produce GABA. Summary of the invention

[0005] In response to the above problems, the present application uses Schisandra chinensis as an inducing factor and Hericium erinaceus as a fermentation fungus for liquid fermentation. It is found that the content of γ-aminobutyric acid increases significantly during the fermentation process, which provides a reference and new approach for the development of Hericium erinaceus into functional food bases and related antioxidant foods.

[0006] On the one hand, the present application provides a method for culturing coral hericium with antioxidant activity, the method comprising: inoculating the coral hericium into a fermentation medium for fermentation culture, wherein the fermentation medium comprises Schisandra chinensis.

[0007] Preferably, the coral hericium contains antioxidant active substances, and the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or γ-aminobutyric acid; preferably, γ-aminobutyric acid.

[0008] The antioxidant activity of the coral hericium obtained by fermentation using this method was improved, especially the content of γ-aminobutyric acid was increased.

[0009] Further, the fermentation medium includes a carbon source, which is selected from one or more of glucose, maltose, corn flour, sucrose, lactose, and mannitol; preferably, lactose; and / or, the fermentation medium also includes a nitrogen source, which is selected from one or more of peptone, yeast extract, ammonium nitrate, beef extract, and yeast extract; preferably, peptone; and / or, the fermentation medium also includes potatoes, magnesium sulfate, dipotassium hydrogen phosphate, and VB1; preferably, 1.6%-2.4% carbon source, 0.1%-0.9% nitrogen source, 10%-50% potato, 0.1%-0.5% magnesium sulfate, 0.1%-0.5% dipotassium hydrogen phosphate, VB10.001%-0.01%, and 0.1%-0.3% schisandra chinensis; more preferably, the fermentation medium includes 1.9% carbon source, 0.3% nitrogen source, 20% potato, 0.15% magnesium sulfate, 0.3% dipotassium hydrogen phosphate, and VB1 0.001%, Schisandra 0.24%, and the balance is water.

[0010] In a preferred embodiment, the fermentation medium comprises: 200 g potato (boiled juice), 19 g lactose, 3 g peptone, 2.4 g Schisandra chinensis, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and 1000 mL water.

[0011] Preferably, the potatoes are potato juice, and the preparation method of the potato juice comprises: peeling the potatoes, cutting them into pieces, adding water, boiling them for 30 minutes, filtering them with gauze to make up the water to 100 mL, and sterilizing them.

[0012] Furthermore, the fermentation culture conditions include: inoculation amount of 0.1%-3%, fermentation temperature of 18°C-32°C, liquid volume of 24%-60%, rotation speed of 100-180rpm, and pH of 4.0-7.0; preferably, the inoculation amount is 1%, the fermentation temperature is 25°C, the liquid volume is 32%, the rotation speed is 140rpm, and the pH is 6.0.

[0013] Preferably, the fermentation time is 1-10 days, more preferably, the fermentation time is 7 days.

[0014] Among them, the inoculation volume refers to the ratio of the volume of the seed liquid transferred in to the volume of the culture liquid after inoculation, and the filling volume refers to the ratio of the volume of the culture medium filled in the flask to the total volume of the flask.

[0015] Furthermore, the coral hericium is coral hericium (Hericium coralloides) RT25, which is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC NO.: 41304.

[0016] In a preferred embodiment, a method for culturing coral hericium with antioxidant activity comprises the following steps:

[0017] Step 1, activating the coral Hericium erinaceus RT25 strain to obtain seed liquid;

[0018] Step 2, inoculating the seed liquid into a fermentation medium for fermentation culture, wherein the fermentation medium comprises: 1.6%-2.4% lactose, 0.1%-0.9% peptone, 10%-50% potato, 0.1%-0.5% magnesium sulfate, 0.1%-0.5% dipotassium hydrogen phosphate, 0.001%-0.01% VB1, 0.1%-0.3% Schisandra chinensis, and the fermentation culture conditions comprise: inoculation amount of 0.1%-3%, fermentation temperature of 18°C-32°C, liquid volume of 24%-60%, rotation speed of 100-180rpm, pH of 4.0-7.0, and fermentation time of 1-10 days.

[0019] Among them, the common method in the art can be used to activate the coral hericium erinaceus strain to obtain the seed liquid.

[0020] In a preferred embodiment, the activation method of the coral hericium erinaceus strain includes: inoculating coral hericium erinaceus blocks into seed liquid culture medium, shaking and culturing at 25° C. and 140 r / min for 7 days.

[0021] Seed liquid culture medium: 20% potato, 2% glucose, 0.3% peptone, 0.3% dipotassium hydrogen phosphate, 0.15% magnesium sulfate, 0.001% VB1, and the balance is water.

[0022] On the other hand, the present application also provides the use of Schisandra chinensis or the method in improving the antioxidant activity of Hericium erinaceus and / or improving the strain quality of Hericium erinaceus.

[0023] Preferably, the coral monkey head is coral monkey head RT25 and / or coral monkey head SY26; more preferably, the coral monkey head is coral monkey head RT25.

[0024] In this application, Schisandra chinensis is used as a substrate for the fermentation of Hericium erinaceus for the first time, and it is found that the Hericium erinaceus obtained by the method can improve the quality of the Hericium erinaceus strain during the fermentation process. The strain quality specifically includes mycelial biomass, ball density, etc., and the ball diameter reaches an appropriate ball density, and can promote the Hericium erinaceus to produce polysaccharides, polyphenols, flavonoids, γ-aminobutyric acid and other metabolites with antioxidant activity.

[0025] In the present application, the fermentation formula is adjusted and optimized to include 200 g of potato (boiled juice), 19 g of lactose, 3 g of peptone, 2.4 g of Schisandra chinensis, 3 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.01 g of VB1, and 1000 mL of water. The fermentation conditions are as follows: inoculation amount 1%, fermentation time 7 d, shaking table speed 140 r / min, pH 6.0, shaking bottle liquid volume 80 mL / 250 mL, and temperature 25 ° C. The average fermentation biomass obtained by the optimized process is 12.258 g, which is 98.24% of the predicted value (12.4780 g).

[0026] In addition, based on the optimization of the Schisandra chinensis fermentation formula, the present application compares the active ingredients (γ-aminobutyric acid, polysaccharides, flavonoids, polyphenols, soluble proteins, triterpenes) and antioxidant activities (total antioxidant capacity, hydroxyl radical (·OH) scavenging rate, DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging rate and superoxide anion (O 2－ ·) clearance rate), and found that the content of γ-aminobutyric acid increased significantly, proving that Schisandra chinensis, as an inducing factor, helps the synthesis of γ-aminobutyric acid, an antioxidant active substance in coral hericium. There has been no precedent for the preparation of γ-aminobutyric acid using coral hericium.

[0027] Therefore, the coral monkey head and its related products obtained by the above method, such as the bacteria, mycelium, fruiting body, fermentation liquid, etc., are rich in antioxidant active substances and have certain antioxidant capacity.

[0028] On the other hand, the present application also provides a coral hericium strain, which is coral hericium (Hericium coralloides) RT25, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and the deposit number is CGMCC NO.: 41304.

[0029] A coral hericium for preparing antioxidant active substances, wherein the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or gamma-aminobutyric acid; preferably, gamma-aminobutyric acid.

[0030] Preferably, the coral monkey head includes coral monkey head and related products, such as:

[0031] A1) Coral Hericium;

[0032] A2) Coral Hericium erinaceus agent;

[0033] A3) Dead Hericium erinaceus suspension;

[0034] A4) Hericium erinaceus metabolites;

[0035] A5) Hericium erinaceus extract;

[0036] A6) Coral Hericium fruiting body;

[0037] A7) Hericium corallii hyphae;

[0038] A8) Hericium cephalosporin.

[0039] On the other hand, the present application also provides coral hericium containing antioxidant active substances prepared by the method, wherein the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or γ-aminobutyric acid; preferably, γ-aminobutyric acid.

[0040] On the other hand, the present application also provides the use of Schisandra chinensis or the described method or the described coral hericium in the preparation of glutamate decarboxylase and / or the preparation of antioxidant active substances; preferably, the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or γ-aminobutyric acid; more preferably, γ-aminobutyric acid.

[0041] On the other hand, the present application also provides the use of Schisandra chinensis as an inducer of growth and / or antioxidant active substances of Hericium erinaceus.

[0042] On the other hand, the present application also provides an inducer of growth and / or antioxidant active substances of Hericium erinaceus, wherein the inducer comprises Schisandra chinensis.

[0043] Preferably, the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or γ-aminobutyric acid; more preferably, γ-aminobutyric acid.

[0044] The present application firstly discovered that Schisandra chinensis can be used as an inducer of growth and antioxidant active substances of Hericium erinaceus, especially can induce an increase in the content of γ-aminobutyric acid, and improve the ability of Hericium erinaceus to autonomously enrich γ-aminobutyric acid by increasing the expression of glutamate decarboxylase activity.

[0045] On the other hand, the present application also provides a coral hericium cultured by the method described or an antioxidant product of the coral hericium.

[0046] The antioxidant product contains antioxidant active substances, and the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or γ-aminobutyric acid; more preferably, γ-aminobutyric acid.

[0047] The product may include excipients, which may be appropriate solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc.

[0048] The product of the present application can be prepared by a general method, wherein one or more diluents or carriers can be added, such as pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions, etc.

[0049] On the other hand, the present application also provides the use of the coral hericium cultured by the method or the coral hericium or the antioxidant product in anti-oxidation.

[0050] The present application also provides the use of the coral hericium cultured by the method or the coral hericium or the antioxidant product in antioxidant activities for purposes other than disease treatment and diagnosis.

[0051] The present invention has the following beneficial effects:

[0052] 1. This application uses Schisandra chinensis as a substrate for the first time to carry out bidirectional fermentation with Hericium erinaceus to obtain Hericium erinaceus rich in antioxidant active ingredients and its related products.

[0053] 2. In this application, the optimal fermentation process of coral hericium was obtained by adjusting and optimizing the fermentation formula and fermentation conditions. The obtained coral hericium strain has the advantages of high ball density, good dispersion, good uniformity of ball diameter, thick hyphae, more lock-like unions, dense and white hyphae, fast primordium formation, large number, large fruiting body, coral shape, soft thorns and fleshy texture.

[0054] 3. The coral hericium and its fermentation liquid and other related products prepared by the fermentation method of the present application contain rich antioxidant active ingredients, especially γ-aminobutyric acid, which provides a new biological source and green biofactory for γ-aminobutyric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0056] Figure 1 It is the gel electrophoresis diagram of PCR products;

[0057] Figure 2 It is a phylogenetic reverse tree diagram;

[0058] Figure 3 This is a schematic diagram of the fermentation substrate screening experiment results;

[0059] Figure 4 This is a statistical chart showing the effects of different fermentation substrates on bacterial quality;

[0060] Figure 5 This is a schematic diagram of the effects of different carbon sources on liquid bacterial pellets;

[0061] Figure 6 This is a statistical chart showing the effects of different carbon sources on bacterial quality;

[0062] Figure 7 This is a schematic diagram of the effects of different nitrogen sources on liquid bacterial pellets;

[0063] Figure 8 This is a statistical chart showing the effects of different nitrogen sources on bacterial quality;

[0064] Fig. 9 This is a statistical chart showing the effect of different peptone concentrations on mycelial biomass;

[0065] Fig.10 This is a statistical chart showing the effect of different Schisandra concentrations on mycelial biomass;

[0066] Fig.11 This is a statistical chart showing the effect of different lactose concentrations on the fermentation of coral hericium;

[0067] Fig.12 Response surface diagram and contour diagram for the pairwise interaction of each factor;

[0068] Fig.13 This is a statistical chart showing the effect of different inoculation amounts on strain quality;

[0069] Fig.14 This is a statistical chart showing the effect of different pH on the quality of bacterial strains;

[0070] Fig.15 This is a statistical chart showing the effect of different liquid volumes on the quality of bacterial strains;

[0071] Fig.16 This is a statistical chart showing the effects of different fermentation temperatures on strain quality;

[0072] Fig.17 This is a statistical chart showing the effect of different shaker speeds on strain quality;

[0073] Fig.18 This is a comparison chart of bacterial growth between the experimental group and the control group;

[0074] Fig.19 This is a pie chart of the active ingredients of Hericium erinaceus mycelium and fruiting body.

[0075] Deposit of biological materials:

[0076] A strain of Hericium coralloides RT25 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 20, 2024, with the deposit number CGMCC NO.: 41304, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences. DETAILED DESCRIPTION

[0077] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0078] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0079] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0080] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in microbiology, biochemistry, analytical chemistry, cell culture, and related fields conventional in the technical field.

[0081] Test strains:

[0082] Hericium coralloides RT25 was deposited in the General Microbiology Center of China Microorganism Culture Collection on May 20, 2024, with the deposit number CGMCC NO.: 41304. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

[0083] The control strain Hericium militaris SY26 was purchased from Shenyang Shanhai Nongren Food Trading Co., Ltd.

[0084] The culture medium involved in the following embodiments includes:

[0085] Seed liquid culture medium: 200g potato (boiled juice), 20g glucose, 3g peptone, 3g dipotassium hydrogen phosphate, 1.5g magnesium sulfate, 0.01g VB1, natural pH, 1000mL distilled water. Sterilize at 121℃ for 30min.

[0086] Method for activation of Hericium erinaceus strain: Use a 9mm hole puncher to punch holes in the plate, and inoculate 3 bacterial blocks in each bottle of seed liquid culture medium. After inoculation, place it in a shaking culture at 25℃ and 140r / min for 7 days to obtain seed liquid.

[0087] Basic fermentation medium (1L): 20g glucose, 3g peptone, 200g potato (boiled juice), 3g dipotassium hydrogen phosphate, 1.5g magnesium sulfate, 0.01g VB1, and the balance is water.

[0088] Bag cultivation medium: 80% broadleaf sawdust, 20% wheat bran, 1% each of gypsum and brown sugar, seasoned to 65% moisture content, natural pH.

[0089] Bag cultivation method: Use bag cultivation medium to avoid light and let the fungus grow for about 37 days until the fungus bag is full. Move the fungus room and place the mushrooms in a four-temperature zone constant temperature and humidification incubator for fruiting. The bag mouth is open for fruiting. The temperature is controlled at 18℃-20℃. Ventilate twice a day. Harvest when the length of the mushroom spines of the coral hericium fruiting body is more than 0.5cm and the spores have not yet been ejected in large quantities.

[0090] Cultivation methods of coral hericium hyphae, primordium and fruiting bodies:

[0091] Step 1, activating the coral hericium and then fermenting and culturing it to obtain liquid strains (or mycelium);

[0092] Step 2: The mycelium obtained by fermentation is rinsed with sterile water, filtered through gauze, dried at a constant temperature of 55°C to constant weight, and crushed through an 80-mesh sieve to obtain a mycelium powder sample for later use; the liquid strain is inoculated into a bag cultivation medium, cultured in a four-temperature zone constant temperature humidification incubator, the primordium and fruiting body are collected, dried at a constant temperature of 55°C to constant weight, crushed through an 80-mesh sieve to obtain a primordium sample and a fruiting body sample, and set aside.

[0093] Among them, coix seed, Fritillaria thunbergii, Poria cocos, licorice, wheat bran, black bean, mulberry leaf, astragalus, Schisandra chinensis, Schisandra chinensis, and Panax notoginseng were all purchased from Chaoyang People's Kangtai Pharmacy (Northern Store); magnesium sulfate, potassium dihydrogen phosphate, anhydrous sodium sulfite, PDA potato agar medium, etc. were all purchased from Beijing Aoboxing Biotechnology Co., Ltd.; phenol, salicin, folin-phenol, and ethanol (analytical grade) were all purchased from Tianjin Komio Chemical Reagent Co., Ltd.; acid buffered saline solution (PBS, pH 7.8) was purchased from Thermo Fisher Scientific (China) Co., Ltd.; hydroxyl radical scavenging ability kit, DPPH free radical scavenging rate kit, and superoxide anion scavenging ability kit were all purchased from Suzhou Greis Biotechnology Co., Ltd.

[0094] The following embodiments involve the following instruments:

[0095] BHC-1300IIA / B2 biological safety cabinet (Shanghai Lishen Scientific Instrument Co., Ltd.); vertical constant temperature shaker (Shanghai Shiping Experimental Equipment Co., Ltd.); KQ-50DA CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 723N visible spectrophotometer (Shanghai Yidian Analytical Instrument Co., Ltd.).

[0096] The detection method involved in the following embodiments includes:

[0097] The determination of strain quality includes the determination of ball diameter, mycelial biomass and ball density.

[0098] Determination of the diameter of bacterial balls: According to the ratio of the number of bacterial balls in each culture medium, 10 bacterial balls are randomly selected and arranged in a long row, and the total length is measured. Repeat 3 times and calculate the average value.

[0099] Determination of mycelial biomass: Determination by differential method. Filter the cultured liquid strain, collect all the bacterial balls in the 250mL shake flask (dry to constant weight before weighing the filter paper), rinse with clean water 3 times, place in a constant temperature drying oven at 60℃ to dry to constant weight, and calculate the average value.

[0100] Determination of bacterial ball density: shake the liquid culture medium from each repeated shaking bottle and place it in a culture dish, count the number of bacterial balls, repeat 3 times and calculate the average value.

[0101] Determination of active ingredient content:

[0102] The polysaccharide content was determined by the phenol-sulfuric acid method, and the polysaccharide standard curve equation was: y = 6.1373x + 0.0284, R 2=0.9947. 0.01 mL of fermentation sample solution was added with 1.99 mL of pure water. The determination method was carried out according to the polysaccharide determination method optimized by Xie Cunyi et al. The polysaccharide content in the fermentation sample solution was calculated according to the standard curve equation and gradient dilution (Reference: Xie Cunyi, Li Jianmei, Guo Lingling, et al. Comparison and optimization of determination methods for extracellular polysaccharide content in mulberry linterus fermentation solution [J]. Edible Fungi, 2022, 44(6): 69-73.).

[0103] The flavonoid content was determined by aluminum nitrate-sodium nitrite colorimetry, and the flavonoid standard curve equation was: y = 2.6125x + 0.0466, R 2 =0.9995. 3 mL of fermentation sample solution was placed in a 25 mL graduated tube, and 2.0 mL of 70% ethanol was added and shaken; 1.0 mL of 5% sodium nitrite solution was added and shaken, and allowed to stand for 6 min; 1.0 mL of 10% aluminum nitrate solution was added and shaken, and allowed to stand for 6 min; 10 mL of 4% sodium hydroxide solution was added, and allowed to stand for 10 min, and then diluted to 25 mL with 70% ethanol, and the absorbance value was measured at 510 nm, and the flavonoid content in the fermentation sample solution was calculated according to the standard curve equation and gradient dilution.

[0104] The polyphenol content was determined by a modified method based on the reference literature, using spectrophotometry, with gallic acid concentration as the horizontal axis and absorbance D as the horizontal axis. 750 As the ordinate, the standard curve equation is: y = 4.379x-0.0059, R 2 =0.9962. Add 120 μL of water to 80 μL of fermentation sample solution and mix well. Add 1 mL of foline phenol and react for 5 min. Then add 0.8 mL of 7.5% Na2CO3. Then add 3 mL of pure water and let stand at 25 °C for 30 min. Measure the absorbance of the fermentation sample solution at 765 nm. Calculate the polyphenol content in the fermentation sample solution according to the standard curve equation and gradient dilution (References: Li Jianmei, Zhu Wanqin, Chai Linshan, et al. Optimization of Cordyceps militaris polyphenol extraction process and its antioxidant activity [J]. Food Research and Development, 2023, 44(8): 111-117.).

[0105] The content of triterpene compounds was detected by the vanillin-glacial acetic acid-perchloric acid method: sample treatment, fermentation broth was centrifuged at 8000×g for 5 min, the supernatant was discarded, an appropriate amount of isopropanol was added to the mycelium precipitate, heated to reflux extraction for 2 h, the filtrate was collected, the solvent was removed by vacuum distillation, and then dried to constant weight at 60°C; about 0.5 g of the fruiting body sample was weighed and put into a mortar, 2.5 mL of pH 7.0 phosphate buffer was added and ground into a homogenate, and isopropanol was refluxed for extraction for 2 h, the filtrate was collected, the solvent was removed by vacuum evaporation, and then dried to constant weight at 60°C. 10.4 mg of the standard betulin was weighed, dissolved in chloroform, and diluted to a 25 mL volumetric flask. 0, 0.25 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, and 4.0 mL of the mother solution were respectively taken in a 10 mL volumetric flask, diluted to a constant volume with chloroform, and 0.2 mL was taken in a test tube. After evaporation, 0.3 mL, 5% vanillin-glacial acetic acid solution and 1 mL of perchloric acid were added. After shaking, the mixture was placed in a 60°C water bath for 45 min, then cooled to room temperature in an ice-water bath, and 5 mL of anhydrous acetic acid was added. After fully shaking, the absorbance was measured at 548 nm to obtain the standard curve equation y=0.0022x+0.0687, R 2 =0.9991. 0.2mL of the extracted sample solution was placed in a test tube. The determination method was the same as the standard curve. The triterpene content was calculated based on the absorbance value and the standard curve equation.

[0106] Determination of γ-aminobutyric acid in samples: add 0.5mL borate buffer (0.2mol / L, pH10.0), 1mL 6% phenol, 1mL NaClO solution (active chlorine is 8%-12%) to 0.5mL of sample extract supernatant, mix well and put in boiling water bath for 10min, take out and immediately put in ice bath for 20min, after the solution turns blue-green, add 2.0mL of 60% ethanol solution and it can be used for determination (using the reagent solution without GABA standard solution as reference). Use 1cm colorimetric dish, scan at wavelength 645nm, obtain absorbance value, and calculate γ-aminobutyric acid concentration using standard curve equation.

[0107] Determination of γ-aminobutyric acid: Reference: Shi Xiaofeng, Luan Guangzhong, Cao Wanxin, Colorimetric determination of γ-aminobutyric acid in traditional fermented bean products, Chinese Condiments, 2008 (3), 76-81. Standard curve determination: Accurately weigh 2500.000 mg of γ-aminobutyric acid in a 500 mL volumetric flask and dissolve it in ultrapure water to make a 5.00 mg / mL standard solution. When using, accurately prepare 0, 0.05, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00 mg·mL -1 The GABA standard solution series was obtained to obtain the standard curve equation y = 0.9901x + 0.1337, R 2 =0.9996, where x is the concentration of γ-aminobutyric acid (mg / mL) and y is the absorbance value.

[0108] Determination of soluble protein content: weigh about 0.5g of sample and put it into a mortar, add 2.5mL pH 7.0 phosphate buffer and grind it into a homogenate, then transfer it to a centrifuge tube, centrifuge it at 8000r / min for 5min, discard the precipitate and obtain the extract to be tested. Preparation of standard curve: take 6 clean test tubes numbered 1, 2, 3, 4, 5, 6, with a mother liquor concentration of 0.5mg / mL, 0, 0.02mL, 0.04mL, 0.06mL, 0.08mL, 0.1mL, then add 5.0mL Coomassie Brilliant Blue G-250 reagent, shake and mix, place it for 2min and then compare the color at 600nm wave, record the optical density value OD measured in each tube, and use the standard protein content (mg / mL) as the horizontal coordinate and the absorbance as the vertical coordinate to draw a standard curve (y=5.8086x+0.0322, R 2 =0.9905). Then, 0.1 mL of the sample extract was added to the test tubes respectively, 0.1 mL of pH 7.0 phosphate buffer was added to the blank control tube, 5.0 mL of Coomassie Brilliant Blue G-250 reagent was added to each tube, and 0.9 mL of distilled water was shaken well. After 2 minutes, the optical density value OD was colorimetrically determined at a wavelength of 600 nm, and the content of soluble protein was calculated by regression equation.

[0109] Determination of free radical scavenging rate:

[0110] The antioxidant activity of the fermentation broth was expressed by the free radical scavenging rate. The DPPH free radical scavenging rate, superoxide anion scavenging rate, and hydroxyl free radical scavenging rate were determined by referring to the method in the article by Xu Bin et al. In the free radical scavenging rate determination of this experiment, the sample solutions were all original solutions and no dilution was required (References: Xu Bin, Zhou Yongkang, Li Huixing, et al. Analysis of the dose-effect relationship between the antioxidant activity of pupa drug fungus and the active ingredients [J]. Food Industry Science and Technology, 2021, 42(2): 250-255.).

[0111] The DPPH free radical scavenging rate of the test sample solution was tested according to the kit instructions. The DPPH free radical scavenging rate of the test sample solution was calculated according to formula (1).

[0112] DPPH free radical scavenging rate = [1-(D 测定 -D 对照 )÷D 空白 ]×100%(1)

[0113] In the formula, D 测定 D is the absorbance value of the mixed solution of sample and DPPH; 对照 D is the absorbance value of the mixed solution of sample and anhydrous ethanol; 空白 It is the absorbance value of the mixture of DPPH and anhydrous ethanol.

[0114] The superoxide anion clearance rate of the test sample was determined according to the kit instructions, and the superoxide anion clearance rate of the test sample was calculated according to formula (2):

[0115] Superoxide anion clearance rate = [1-(D 测定 -D 对照 )÷D 空白 ]×100%(2)

[0116] In the formula, D 测定 is the absorbance value of the measuring tube; D 对照 is the absorbance value of the control tube; D 空白 is the absorbance value of the blank tube

[0117] The hydroxyl radical scavenging rate of the test sample was determined according to the kit instructions, and the hydroxyl radical scavenging rate of the test sample was calculated according to formula (3):

[0118] Hydroxyl radical scavenging rate = [D 空白－ (D 测定 -D 对照 )]÷D 空白 ×100%(3)

[0119] In the formula, D 测定 is the absorbance value of the measuring tube; D 对照 is the absorbance value of the control tube; D 空白 is the absorbance value of the blank tube.

[0120] The total antioxidant capacity was determined using the T-AOC assay kit and the total antioxidant capacity of the test sample was calculated according to formula (4):

[0121] Total antioxidant capacity / (μmol Trolox / g)=0.3×(△A+0.029)÷W×D(4)

[0122] In the formula: △A = A blank - (A determination - A control), where A determination is the absorbance of the sample and ABTS working solution, A control is the absorbance of the mixed solution of the sample and PBS, and A blank is the absorbance of the mixed solution of ABTS working solution and PBS; W is the sample mass in g; D is the dilution factor of the sample.

[0123] All results were expressed as mean ± standard deviation. Excel software was used for data statistics and analysis, with P < 0.05 indicating a significant difference and P < 0.01 indicating an extremely significant difference. Design-Expert 11.0 software was used for model regression analysis and response surface interaction analysis.

[0124] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion exchange water, double distilled water, high-purity water, and purified water.

[0125] In the following examples, unless otherwise specified, % means wt %, i.e. weight percentage.

[0126] Example 1 Bacterial species identification and strain preservation

[0127] In this implementation, a new strain of Hericium erinaceus was obtained through screening and named RT25. Molecular identification and phylogenetic study of the Hericium erinaceus RT25 were conducted, including genomic DNA extraction, PCR product sequencing and phylogenetic analysis of the Hericium erinaceus RT25. After purification, unidirectional sequencing was performed, and the rDNA ITS sequence of the test strain was measured using the GenBank nucleic acid sequence database to establish a phylogenetic reverse tree. Among them, the electrophoresis diagram of the PCR product is shown in Figure 1 The reverse phylogenetic tree is shown in Figure 2 shown.

[0128] The test strain Hericium coralloides RT25 was submitted to Shanghai Sangon Biotechnology Co., Ltd. for strain identification, and the strain was finally identified as Hericium (Hericium erinaceus), Hericium coralloides. Currently, the strain Hericium coralloides RT25 has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 20, 2024, with the deposit number CGMCC NO.: 41304.

[0129] Example 2 Fermentation substrate screening experiment

[0130] Different fermentation substrates have different effects on the strain quality of Hericium erinaceus RT25. In this embodiment, the fermentation substrate of Hericium erinaceus RT25 was screened, and the specific method includes:

[0131] The fermentation substrates Coix chinensis Tod., Fritillaria thunbergii, Wolfiporia cocos, Glycyrrhiza uralensis, Wheat bran, Glycinemax, Folium Mori, Astragalus mongholicus Bunge, Schisandra chinensis, and Panax notoginseng were crushed by a grinder, sieved through a 60-mesh sample sieve, and set aside. 0.2 g·100 mL -1Add them into the basic fermentation medium respectively, set the pH to natural, sterilize at 121℃ for 30min to obtain the substrate medium. The blank control medium is the basic fermentation medium.

[0132] The above substrate culture medium and basic fermentation culture medium were used to ferment Hericium erinaceus RT25, and the culture process was as follows: step 1, activating Hericium erinaceus RT25 to obtain seed liquid; step 2, inoculating the seed liquid with an inoculation amount of 1% into the above substrate culture medium containing different fermentation substrates for fermentation culture, and liquid culture was carried out at 25°C, 140r / min, and natural pH. After 7 days, the quality of the strains of each formula (mycelial biomass, ball diameter, and ball density) was measured. The results are as follows Figure 3 , as shown in Table 1, the content of Table 1 is used to make Figure 4 .

[0133] Table 1 Effects of different fermentation substrates on strain quality

[0134]

[0135] like Figure 3 , Table 1 and Figure 4 As shown, Schisandra chinensis can significantly promote the growth of strains, while Fritillaria thunbergii, Coix seeds and Panax notoginseng have significant inhibitory effects. Among them, when Schisandra chinensis is used as a fermentation substrate, the mycelial biomass is the highest, significantly higher than other substrates, the ball diameter is not large (the ball diameter does not block the muzzle during inoculation, and can ensure sufficient mycelial volume), the ball density is significantly higher than other substrates, the ball density is moderate, and it is suitable for production as a liquid strain. Therefore, Schisandra chinensis is selected as a fermentation substrate for subsequent fermentation.

[0136] Example 3 Carbon source test

[0137] In this embodiment, a single factor experiment was designed to screen the carbon source. Liquid culture medium was prepared according to different carbon sources. Liquid culture was carried out with an inoculum size of 1%, 25°C, 140r / min, and natural pH. After 7 days, the quality of Hericium erinaceus RT25 strains (mycelial biomass, ball diameter, and ball density) of each formula was measured. The rest of the culture method was the same as in Example 2. The test results are shown in FIG. Figure 5 As shown in Table 2, the graph obtained from Table 2 is Figure 6 .

[0138] The formula of the liquid culture medium (1 L) is: 20 g carbon source, 3 g peptone, 2 g Schisandra chinensis, 200 g potato (boiled juice), 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance is water.

[0139] Carbon sources include glucose, maltose, corn flour, sucrose, lactose, and mannitol.

[0140] The blank control medium was used as the basic fermentation medium.

[0141] Table 2 Effect of different carbon sources on strain quality

[0142]

[0143] The screening results of different carbon sources are shown in Figure 5 , Table 2 and Figure 6 , among which lactose as carbon source has the highest mycelial biomass, which is 8.208g / L, significantly higher than other carbon sources, followed by maltose; when lactose is used as carbon source, although the diameter of the ball is not the smallest, it will not block the muzzle during inoculation, and can ensure sufficient mycelial volume, with a ball density of 205 / 100mL, significantly higher than maltose and sucrose, and the ball density is moderate, which is suitable for production as liquid strains. Therefore, lactose can be used as the best carbon source for the production of coral hericium liquid strains.

[0144] Example 4 Nitrogen source test

[0145] The single factor experiment was designed in the same way as in Example 3 to screen the nitrogen source. Liquid culture medium was prepared according to different nitrogen sources. Liquid culture was carried out at 1% inoculation amount, 25°C, 140r / min, and natural pH. After 7 days, the quality of coral hericium erinaceus RT25 strains (mycelial biomass, ball diameter, and ball density) of each formula was measured. The rest of the culture method was the same as in Example 2. The test results are as follows: Figure 7 As shown in Table 3, the graph obtained from Table 3 is Figure 8 .

[0146] The formula of the liquid culture medium (1 L) is: 3 g nitrogen source, 20 g glucose, 200 g potato (boiled juice), 2 g Schisandra chinensis, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance is water.

[0147] Types of nitrogen sources include peptone, yeast extract powder, ammonium nitrate, tryptone, beef extract, and yeast extract.

[0148] The blank control medium was used as the basic fermentation medium.

[0149] Table 3 Effect of different nitrogen sources on strain quality

[0150]

[0151] The results of the screening of nitrogen sources for the liquid strain of Hericium erinaceus are shown in Figure 7 , Table 3, Figure 8When peptone was used as the nitrogen source, the mycelial biomass was 7.144 g / L, which was significantly higher than other nitrogen sources. The ball diameter was 1.127 mm, which did not block the muzzle during the inoculation process and could ensure sufficient mycelial mass. The ball density was 579 / 100 mL, which was suitable as a nitrogen source for liquid strains. The second was yeast extract powder, whose mycelial biomass was 5.941 g / L. When yeast extract powder was used as the nitrogen source for liquid strains, the ball density was too low and the ball diameter was too large. Therefore, peptone was selected as the best nitrogen source for coral hericium liquid culture medium. When ammonium nitrate inorganic nitrogen was selected as the nitrogen source, the mycelial biomass and ball density were both low, which was not suitable as a nitrogen source for liquid strains.

[0152] Example 5 Test on the Optimal Addition Amount of Fermentation Medium

[0153] In this example, a single factor experiment of addition amount was conducted for the optimal culture medium components (peptone, potato, lactose, magnesium sulfate, potassium dihydrogen phosphate, VB1, Schisandra chinensis) obtained in Examples 2-4, and coral Hericium erinaceus RT25 was cultured with culture medium of different concentrations, as shown in Table 4, and liquid culture was carried out with 1% inoculation amount, 25°C, 140r / min, and natural pH. After 7 days, the strain quality (mycelial biomass, ball diameter, ball density) of each formula and the content of γ-aminobutyric acid in the mycelium were measured. The rest of the culture method was the same as in Example 2. The test results are shown in Table 4. The plot of Table 4 shows Figure 9-11 .

[0154] Among them, the formula of the single-factor experimental fermentation medium (1L) with added peptone is: 1-9g peptone, 200g potato (boiled juice), 20g glucose, 2g Schisandra chinensis, 1.5g magnesium sulfate, 3g dipotassium hydrogen phosphate, 0.01g VB1, and 1L water.

[0155] The formula of the single-factor experimental fermentation medium (1L) for lactose addition is: 16-24g lactose, 200g potato (boiled juice), 3g dipotassium hydrogen phosphate, 1.5g magnesium sulfate, 3g peptone, 2g Schisandra chinensis, 0.01g VB1, and the balance is water.

[0156] The formula of the single-factor experimental fermentation medium (1L) of Schisandra added was: 1-3g Schisandra, 3g peptone, 200g potato (boiled juice), 20g lactose, 1.5g magnesium sulfate, 3g potassium dihydrogen phosphate, 0.01g VB1, and the balance was water.

[0157] Table 4 Single factor test of fermentation nutrient composition

[0158]

[0159]

[0160] From Table 4 and Figure 9-11 The results showed that the optimal carbon-nitrogen ratio fermentation medium (1L) formula was 3g peptone, 18g lactose, 2.5g Schisandra chinensis, 200g potato (boiled juice), 3g dipotassium hydrogen phosphate, 1.5g magnesium sulfate, 0.01g VB1, and the remainder was water.

[0161] Furthermore, it was found for the first time in this example that the addition of Schisandra chinensis could cause a change in the content of γ-aminobutyric acid in the strain.

[0162] Example 6 Fermentation recipe optimization

[0163] Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter of the central nervous system, which has good water solubility and thermal stability. It has been confirmed that GABA, as a small molecular weight non-protein amino acid, has edible safety and can be used in the production of foods such as beverages. At present, studies have shown that taking a certain amount of GABA has physiological effects such as improving body sleep quality and lowering blood pressure. Comprehensive comparison, the active ingredient γ-aminobutyric acid is significantly correlated with antioxidant activity, and embodiment 5 preliminarily proves that γ-aminobutyric acid is a coral hericium antioxidant active substance. In this embodiment, the coral hericium erinaceus RT25 fermentation medium is further subjected to single factor test and response surface method optimization using the dual index parameters of fermentation biomass and γ-aminobutyric acid content, in order to provide a reference for the development of coral hericium erinaceus fermentation products and improving their bioavailability.

[0164] According to the single factor test results in Example 2-5, the fermentation test was carried out according to the response surface factor level design table 5, and the weighted comprehensive score was used as the response value (Y). Combined with the single factor test results, the maximum value of the fermentation biomass 12.772 was counted as 50 points, and its corresponding score was (N1 / 12.772)×50, and the maximum score of γ-aminobutyric acid content 28.778 points was counted as 50 points, and its corresponding score was (N2 / 28.778)×50. Comprehensive score=(N1 / 12.772)×50+(N2 / 28.778)×50, where N1 is the fermentation biomass, N2 is the γ-aminobutyric acid content, and the measurement results and comprehensive score results are shown in Table 6.

[0165] The results of variance analysis of the regression model are shown in Table 7. The regression equation between the comprehensive score (Y1) and lactose (X1), peptone (X2), and Schisandra chinensis (X3) was calculated as follows:

[0166] Y1=89.64-7.9X1+1.39X2-0.131X3-0.21X1X2+6.53X1X3+0.8065X2X3-16.05X1 2 -20.57X2 2 -16.58X

[0167] 3 2 (5)

[0168] The coefficient of determination R of the model 2 =0.9128, Corrected determination coefficient R 2 adj = 0.8007, indicating that 80.07% of the changes in the comprehensive score can be predicted by this model. The specific results are shown in Table 7. From Table 7, we can see that the model F = 8.14, P < 0.01, indicating that the model is significant, the lack of fit item F = 1.63, P = 0.3174 > 0.05, indicating that the lack of fit test is not significant, the equation fit is good, and the difference between the model and the test value is small. According to the F values ​​of each item in Table 7, we can see that: A 2 , B 2 , C 2 The influence of etc. on the comprehensive score reached an extremely significant level, and A reached a significant level. The order of the influence on the comprehensive score can also be judged from the size of the F value: lactose>peptone>Schisandra chinensis.

[0169] Table 5 Response surface factor levels

[0170]

[0171] Table 6 Response surface experimental design results

[0172]

[0173]

[0174] Table 7 Analysis of variance of regression model

[0175]

[0176] Note: * indicates that the correlation reaches a significant level (P < 0.05); ** indicates that the correlation reaches an extremely significant level (P < 0.01)

[0177] The 3D response surface illustrates the interaction between the dependent variable and the two test variables when the other factors are fixed at zero level. The interaction surface plot of equation (5) was made using Design-Expert 11.0 software, and the result was Fig.12, after fixing the value of one of the factors A, B, and C in the regression model, the interactions between the two factors formed a convex surface opening downward; within the value range of A, B, and C, the comprehensive score Y increased first and then decreased with the increase of concentration, and there was a maximum point; from the contour map, it can be seen that the slope of the response surface of the interaction between lactose and Schisandra chinensis (AC) was steep, and the effect on the comprehensive score Y was significant; the interaction between lactose concentration and peptone concentration (AB), and between peptone concentration and Schisandra chinensis concentration (BC) had no significant effect on Y, and the interaction effect was AC>BC>AB, which was consistent with the results of variance analysis.

[0178] Taking the first-order partial derivative of equation (5), we get:

[0179]

[0180] After calculation, when the response value is the maximum value, the optimum values ​​of ABC are 19.054g / L, 3.068g / L, and 2.392g / L, respectively. Under this condition, the maximum value of the comprehensive score is 90.644. The optimized fermentation process after adjustment is: 200g potato (boiled juice), 19g lactose, 3g peptone, 2.4g Schisandra chinensis, 3g dipotassium hydrogen phosphate, 1.5g magnesium sulfate, 0.01g VB1, 1000mL water, 1% inoculation, 7d fermentation time, 140r / min shaking speed, pH 6.0 (natural), 80mL / 250mL shaking bottle liquid volume, 25℃ temperature. The optimized process was used and the experiment was repeated three times. The average fermentation biomass was 12.258g / L, and the average γ-amino acid content was 28.087mg / g, indicating that the model is feasible and can provide a reference for the fermentation process of coral hericium.

[0181] Example 7 Environmental Conditions Test

[0182] The optimal fermentation medium formula obtained in Example 6 was used to design a single factor experiment with 5 factors and 5 levels, including inoculation amount, fermentation temperature, liquid volume, rotation speed, and pH, as shown in Table 8 below. The remaining culture methods were the same as in Example 2, and each group of experiments was repeated 3 times. The strain quality was determined, and the results were prepared according to Table 8. Figure 13-17 .

[0183] Table 8 Single factor test of coral hericium erinaceus RT25 fermentation environmental conditions

[0184]

[0185] As shown in Table 8 and Figure 13-17 Based on the experimental results of environmental conditions, combined with the mycelial biomass, ball density and average ball diameter, the appropriate inoculation amount was selected as 1%, the fermentation pH was 6, the shake flask liquid volume was 80mL / 250mL, the fermentation temperature was 25℃, and the shaker speed was 140r / min.

[0186] Example 8 Fermentation formula verification

[0187] The optimized liquid fermentation medium obtained in Example 6 and the optimal fermentation conditions obtained in Example 7 were used for fermentation and fruiting tests. The coral Hericium erinaceus fermentation group without the addition of Schisandra chinensis was used as the control group, the fermentation medium with the addition of Schisandra chinensis was used as the test group, the coral Hericium erinaceus fruiting body obtained by inoculating the fermentation liquid strain without the addition of Schisandra chinensis was used as the control group, and the coral Hericium erinaceus fruiting body inoculated with the Schisandra optimized medium fermentation liquid strain was used as the test group. The strain RT25 was used as an example to test the strain quality, and the mycelium was collected and cultivated in bags to obtain primordia and fruiting bodies. The fermentation mycelium state, fruiting mycelium growth rate, fruiting mycelium full growth days, primordium formation days, fruiting body fresh weight, and fruiting body dry weight of the strain were recorded. The results are shown in Table 9. Fig.18 shown.

[0188] Control group: The strain RT25 was inoculated into the control group culture medium (200 g potato (boiled juice), 19 g lactose, 3 g peptone, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance was water, a total of 1 L), the inoculation amount was 1%, the fermentation time was 7 days, the shaking table speed was 140 r / min, the pH was 6.0 (natural), the shaking bottle liquid volume was 80 mL / 250 mL, and the temperature was 25°C.

[0189] Experimental group: The strain RT25 was inoculated into the experimental group culture medium (200 g potato (boiled juice), 19 g lactose, 3 g peptone, 2.4 g Schisandra chinensis, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance was water, a total of 1 L), with an inoculation size of 1%, a fermentation time of 7 days, a shaking table speed of 140 r / min, a pH of 6.0 (natural), a shaking bottle liquid volume of 80 mL / 250 mL, and a culture temperature of 25°C.

[0190] From Table 9, Fig.18 It can be seen that the fermentation liquid of the experimental group has more uniform bacterial balls and a larger density than that of the control group. The mycelium growth rate, fruiting body size and fruiting body weight of the experimental group are significantly improved compared with those of the control group, indicating that Schisandra chinensis has a promoting effect on the growth of coral hericium. The fermentation formula is highly feasible and has broad application prospects.

[0191] Table 9 Comparison between the control group and the experimental group

[0192]

[0193] Example 9: Mining of antioxidant active ingredients

[0194] In this example, the effects of Schisandra chinensis and strain RT25 on the antioxidant active components in mycelium and fruiting bodies and their changes were further explored, and strain SY26 was used as a control.

[0195] Strain RT25 experimental group: The strain RT25 was inoculated into the experimental group culture medium (200 g potato (boiled juice), 19 g lactose, 3 g peptone, 2.4 g Schisandra chinensis, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance was water, a total of 1 L), with an inoculation size of 1%, a fermentation time of 7 days, a shaking table speed of 140 r / min, a pH of 6.0 (natural), a shaking bottle liquid volume of 80 mL / 250 mL, and a culture temperature of 25°C.

[0196] Strain RT25 control group: The strain RT25 was inoculated into the control group culture medium (200 g potato (boiled juice), 19 g lactose, 3 g peptone, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance was water, a total of 1 L), the inoculation size was 1%, the fermentation time was 7 days, the shaking table speed was 140 r / min, the pH was 6.0 (natural), the shaking bottle liquid volume was 80 mL / 250 mL, and the temperature was 25°C.

[0197] Strain SY26 experimental group: The strain SY26 was inoculated into the experimental group culture medium (200 g potato (boiled juice), 19 g lactose, 3 g peptone, 2.4 g Schisandra chinensis, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance was water, a total of 1 L), the inoculation size was 1%, the fermentation time was 7 d, the shaking table speed was 140 r / min, the pH was 6.0 (natural), the shaking bottle liquid volume was 80 mL / 250 mL, and the temperature was 25 ° C.

[0198] Strain SY26 control group: The strain SY26 was inoculated into the control group culture medium (200 g potato (boiled juice), 19 g lactose, 3 g peptone, 3 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate, 0.01 g VB1, and the balance was water, a total of 1 L), the inoculation amount was 1%, the fermentation time was 7 d, the shaking table speed was 140 r / min, the pH was 6.0 (natural), the shaking bottle liquid volume was 80 mL / 250 mL, and the temperature was 25 ° C.

[0199] After obtaining fermented mycelium using the above-mentioned fermentation method, the fermented mycelium of the RT25 control group and the experimental group of the above-mentioned strain was inoculated into the bag cultivation medium to obtain the RT25 strain fruiting body control group and the experimental group; the mycelium of the RT26 control group and the experimental group was inoculated into the bag cultivation medium to obtain the RT26 strain fruiting body control group and the experimental group, and then the content of antioxidant active ingredients therein was measured respectively.

[0200] The comparison results of active ingredients and free radical scavenging rates in the mycelium and fruiting bodies of the test strain RT25 and the control strain SY26 are shown in Table 10. This example proves that after adding Schisandra chinensis as a fermentation factor, the active ingredients and free radical scavenging rates in the sample liquid of the test group are improved, indicating that adding Schisandra chinensis as an inducing factor has a certain synergistic effect on the content of active substances and antioxidant activity in the mycelium and fruiting bodies of Hericium erinaceus.

[0201] Pearson correlation analysis was used to determine the correlation between antioxidant active substances and antioxidant activity (reference: Ge Xinhui, Meng Jikun, et al., Analysis of phenolic composition and antioxidant activity of wild edible fungi in Wutai Mountain, Chinese Edible Fungi. 2022, 41(04)) (Table 11), and the percentage of each active substance in the total antioxidant active substance content was calculated ( Fig.19 ), from the correlation analysis in Table 11, it can be seen that the antioxidant capacity of coral hericium mycelium and fruiting body is the result of the synergistic effect of multiple active ingredients, among which γ-aminobutyric acid, polysaccharides, flavonoids, polyphenols and other active ingredients have different correlations with antioxidant capacity: γ-aminobutyric acid is extremely significantly positively correlated with DPPH free radical scavenging rate (P=0.888), and is extremely significantly positively correlated with hydroxyl free radical scavenging rate (P=0.945); polysaccharides are extremely significantly positively correlated with hydroxyl free radical scavenging rate. There was a significant positive correlation between flavonoids and hydroxyl radical scavenging rate (P=0.825), and a significant positive correlation with DPPH free radical scavenging rate (P=0.68). There was a significant positive correlation between polyphenols and DPPH free radical scavenging rate and hydroxyl free radical scavenging rate (P=0.737, P=0.812), and a positive correlation with superoxide anion (P=0.705).

[0202] Table 10 Active ingredients and antioxidant activity of Hericium erinaceus

[0203]

[0204] Table 11 Correlation analysis

[0205]

[0206]

[0207] Note: ***, **, * represent 1%, 5%, 10% significance levels respectively

[0208] Depend on Fig.19Analysis of the increase in γ-aminobutyric acid showed that after the addition of Schisandra chinensis to induce fermentation, the γ-aminobutyric acid content in the active ingredients of RT25 mycelium increased by 21%, while the γ-aminobutyric acid content in the active ingredients of the control strain SY26 mycelium increased by 7%; the percentage of γ-aminobutyric acid in the active ingredients of RT25 fruiting bodies increased by 7%, which was two percentage points higher than that of the control strain SY26 fruiting bodies. Therefore, the experimental strain RT25 can better utilize the Schisandra chinensis fermentation medium than the control strain SY26, and promote the biosynthesis of its antioxidant active substance γ-aminobutyric acid, that is, the Schisandra chinensis induction factor helps the synthesis of the antioxidant active substance γ-aminobutyric acid in coral hericium, and the effect on the strain RT25 is particularly significant.

[0209] Example 10 Determination of glutamate decarboxylase (GAD) activity in coral hericium

[0210] In this example, the reason why Hericium erinaceus is rich in γ-aminobutyric acid was further explored, and the glutamate decarboxylase (GAD) activity was measured in mycelium as an example.

[0211] The specific method is as follows:

[0212] Step 1: Use 250 mL Erlenmeyer flasks for seed culture, with a seed liquid culture medium volume of 100 mL. After inoculating Hericium erinaceus RT25 or SY26, culture at 25° C., 140 rpm for 7 days until the culture medium is full of mycelial balls the size of fish eggs, and culture until the culture liquid is clear, thus obtaining Hericium erinaceus seed liquid.

[0213] Step 2, inoculating the seed liquid of strains RT25 and SY26 into the experimental group culture medium and the control group culture medium, respectively, and fermenting and culturing until the culture medium is full of rice-sized mycelial balls (the specific grouping and culturing process are as follows), thereby obtaining coral hericium mycelium, filtering the coral hericium mycelium with a wire mesh, rinsing it with physiological saline three times, squeezing out water, and obtaining coral hericium mycelium.

[0214] The fermentation medium and fermentation conditions of the strain RT25 experimental group, the strain RT25 control group, the strain SY26 experimental group, and the strain SY26 control group were the same as those in Example 9.

[0215] Extraction of crude enzyme solution: Collect 3 g of coral Hericium erinaceus in step 2, add 30 mL of Mac Ilvaine buffer (pH 4.8, containing 1 mmol / L mercaptoethanol and 0.1 mmol / L pyridoxal phosphate), and disrupt the cells by ultrasonic wave in an ice bath. The disruption conditions are power 800 W, 15 min (working 5 s, interval 15 s), add 0.05 mL of Tween 80 and a little quartz sand, grind in an ice bath for 5 min, centrifuge (8000 r / min) to collect the supernatant, dialyze in Mac Ilvaine buffer (pH 4.8, containing 1 mmol / L mercaptoethanol and 0.1 mmol / L pyridoxal phosphate), and then centrifuge to collect the supernatant as the crude enzyme solution of glutamate decarboxylase (GAD).

[0216] The enzyme reaction experiment includes the following steps:

[0217] Take 2mL GAD crude enzyme solution, 2mL glutamic acid solution (60mmol / L, pH4.8) and 2mL Mac Ilvaine buffer (pH4.8, containing 0.4mmol / L pyridoxal phosphate), mix well and react at 40℃ for 20h, take out and put in ice bath to terminate the reaction. Perform the same operation with GAD crude enzyme solution that has been inactivated (boiling water bath for 10min) as blank.

[0218] The GABA produced by glutamate decarboxylase (GAD) was determined by phenol and sodium hypochlorite colorimetry, and a blue substance was finally generated. The substance had obvious light absorption at 645 nm, and the color depth was proportional to the GABA content, and the GAD activity was then obtained. The results are shown in Table 12.

[0219] Standard curve: y = 2.0976x-0.0059, x is the molar mass of the standard (μmol), y is the absorbance value ΔA. Calculated by the fresh weight of the sample, unit definition: 1 nmol of GABA produced per gram of tissue per minute is defined as one enzyme activity unit.

[0220] GAD(nmol / min / g fresh weight)=[(ΔA+0.0059)÷2.0976]×(V2÷V3)÷(W×V1÷V)÷T×103=397.3×(ΔA+0.0059)÷W

[0221] V——volume of extract, 1mL; V1——volume of sample added to the reaction system, 0.1mL; V2——total volume of the reaction system: 0.5mL; V3——volume of supernatant in the color development stage, 0.2mL; T——reaction time, 30min; W——sample mass, g; molecular weight of standard GABA——103.12.

[0222] Table 12 Glutamate decarboxylase activity in Hericium erinaceus

[0223]

[0224] It can be seen from the results in Table 12 that the use of Schisandra chinensis as an inducing factor to ferment Hericium erinaceus in the present application can increase the expression of glutamate decarboxylase activity in mycelium, primordium, and fruiting body to varying degrees, thereby improving the ability of Hericium erinaceus to autonomously enrich γ-aminobutyric acid.

[0225] in conclusion:

[0226] In recent years, the production scope of coral hericium has gradually expanded from the northeast to the south. With the proposal of the "going out" development strategy, coral hericium has been successfully introduced to Zambia and integrated with tropical plateau supporting industrialization technology. The main active ingredients of coral hericium are polysaccharides, sterols, hericium erinaceus ketones, etc. Cheng Yanfen et al. found that coral hericium polysaccharides can effectively regulate the blood lipid levels of hypercholesterolemic rats; Meng Junlong et al. found that coral hericium is an edible fungus with high protein content, rich minerals, and complete essential amino acids. Its fungal polysaccharides have the effect of enhancing the immune function of mice; Tang Peng et al. found that coral hericium polysaccharides have a regulatory effect on the metabolism of rat liver. At present, there is no report on the correlation analysis of the active ingredients and antioxidant activity of coral hericium-Schisandra chinensis bidirectional liquid fermentation.

[0227] This study was the first to use Schisandra chinensis as a fermentation substrate for coral hericium for bidirectional fermentation, constructed a coral hericium-Schisandra chinensis liquid fermentation system, clarified the antioxidant synergistic principle of Schisandra chinensis fermented by coral hericium, and broke through the characteristic bidirectional liquid fermentation technology of coral hericium-Schisandra chinensis. The fermentation formula was adjusted and optimized to 200g potato (boiled juice), 19g lactose, 3g peptone, 2.4g Schisandra chinensis, 3g dipotassium hydrogen phosphate, 1.5g magnesium sulfate, 0.01g VB1, and the balance was water, a total of 1000mL, and the fermentation conditions were inoculation amount 1%, fermentation time 7d, shaking speed 140r / min, pH 6.0 (natural), shake flask liquid volume 80mL / 250mL, temperature 25℃, and optimized process were used to obtain an average fermentation biomass of 12.258g, which was 98.24% of the predicted value (12.4780g), and the average γ-amino acid content was 28.087mg / g; in terms of active ingredients and antioxidant capacity, the coral hericium erinaceus antioxidant active ingredient γ-aminobutyric acid was excavated, and the coral hericium erinaceus-Schisandra chinensis liquid fermentation system was adopted. The test strain RT25 was able to utilize the Schisandra chinensis fermentation medium better than the control strain SY26, and the Schisandra chinensis induction factor was helpful for the synthesis of the coral hericium erinaceus antioxidant active substance γ-aminobutyric acid, and the effect on the strain RT25 was particularly significant.

[0228] In this study, the coral hericium-schisandra fermentation system achieved a simultaneous increase in the content of active substances and antioxidant activity. The fermentation has "bidirectionality" and excavated the coral hericium antioxidant active substance γ-aminobutyric acid. Under the induction of the coral hericium-schisandra fermentation system, the γ-aminobutyric acid in the mycelium and fruiting body of the coral hericium strain RT25 increased significantly. In the mycelium stage, based on the coral hericium-schisandra fermentation system, high biological activity mycelium was obtained, the coral hericium yield was increased, and it can also be used as a functional oral liquid carrier. In the fruiting body stage, the coral hericium-schisandra fermentation system was inoculated to obtain fruiting bodies rich in antioxidant active substances, thereby providing a new biomaterial for high antioxidant activity and growth-promoting and sleep-promoting biosynthetic preparations. Comprehensive analysis shows that the coral hericium-schisandra fermentation system has broad application prospects.

[0229] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A method for cultivating coral hericium with antioxidant activity, characterized in that: The method comprises: inoculating coral hericium into a fermentation medium for fermentation culture, wherein the fermentation medium comprises Schisandra chinensis.

2. The method according to claim 1, characterized in that The fermentation medium comprises a carbon source, which is selected from one or more of glucose, maltose, corn flour, sucrose, lactose, and mannitol; preferably, lactose; and / or, the fermentation medium further comprises a nitrogen source, which is selected from one or more of peptone, yeast extract, ammonium nitrate, beef extract, and yeast extract; preferably, peptone; and / or, the fermentation medium further comprises potatoes, magnesium sulfate, dipotassium hydrogen phosphate, and VB1; preferably, 1.6%-2.4% of carbon source, 0.1%-0.9% of nitrogen source, 10%-50% of potato, 0.1%-0.5% of magnesium sulfate, 0.1%-0.5% of dipotassium hydrogen phosphate, 0.001%-0.01% of VB1, and 0.1%-0.3% of Schisandra chinensis; more preferably, the fermentation medium comprises 1.9% of carbon source, 0.3% of nitrogen source, 20% of potato, 0.15% of magnesium sulfate, 0.3% of dipotassium hydrogen phosphate, and VB1 0.001%, Schisandra 0.24%, and the balance is water.

3. The method according to claim 1, characterized in that The fermentation culture conditions include: inoculation amount of 0.1%-3%, fermentation temperature of 18°C-32°C, liquid volume of 24%-60%, rotation speed of 100-180rpm, and pH of 4.0-7.0; preferably, the inoculation amount is 1%, the fermentation temperature is 25°C, the liquid volume is 32%, the rotation speed is 140rpm, and the pH is 6.

0.

4. The method according to claim 1, characterized in that: The coral hericium is coral hericium (Hericium coralloides) RT25, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and the deposit number is CGMCC NO.: 41304.

5. Use of Schisandra chinensis or the method according to any one of claims 1 to 4 in improving the antioxidant activity of Hericium erinaceus and / or improving the strain quality of Hericium erinaceus.

6. A coral hericium strain, characterized in that: The coral hericium is coral hericium RT25, which is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and the deposit number is CGMCC NO.: 41304.

7. A coral hericium containing antioxidant active substances prepared by the method according to any one of claims 1 to 4, characterized in that: The antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or γ-aminobutyric acid; preferably, γ-aminobutyric acid.

8. Use of Schisandra chinensis or the method according to any one of claims 1 to 4 or the coral hericium according to claim 6 or 7 in the preparation of glutamate decarboxylase and / or the preparation of antioxidant active substances; preferably, the antioxidant active substances include polysaccharides, flavonoids, polyphenols, triterpenes, soluble proteins and / or γ-aminobutyric acid; more preferably, γ-aminobutyric acid.

9. An antioxidant product comprising the coral hericium erinaceus cultured according to any one of claims 1 to 4 or the coral hericium erinaceus according to claim 6 or 7.

10. Use of the coral hericium cultured according to any one of claims 1 to 4, the coral hericium according to claim 6, or the antioxidant product according to claim 9 in anti-oxidation.

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